Voltammetric reference control system

ABSTRACT

A voltammetric reference control system for generating a reference signal for an electrochemical sensor. The electrochemical sensor may comprise a voltammetric sensor, a potentiometric sensor, an amperometric sensor, an ion selective sensor and/or the like that is designed to be used in solutions, such as water, seawater, saline solutions and/or the like. The voltammetric reference control system generates a reference potential that does not drift and/or does not require calibration during operation of the electrochemical sensor.

This application is a U.S. National Stage of International ApplicationNo. PCT/EP2018/077780, filed Oct. 11, 2018, which claims the benefit ofGreat Britain Patent Application No. 1716652.1 filed on Oct. 11, 2017,Great Britain Patent Application No. 1716660.4 filed on Oct. 11, 2017,and International Application No. PCT/IB2018/000097, filed Feb. 12,2018; the entire disclosures of all are hereby incorporated by referencein their entireties into the present disclosure for all purposes.

BACKGROUND

Embodiments of the present disclosure provide a voltammetric referencecontrol system configured to generate a reference signal potential anelectrochemical sensor. The electrochemical sensor may comprise avoltammetric sensor, a potentiometric sensor, an amperometric sensor, anion selective sensor and/or the like that is configures for operation inlow buffering capacity solutions, such as water, seawater, salinesolutions and/or the like. The voltammetric reference control systemcontrols reference electrode to generate a reference potential that doesnot drift and/or does not require calibration during operation of theelectrochemical sensor, where the reference electrode comprises a redoxchemistry sensitive to proton concentration and the reference electrodeis configured to set a pH of the low buffering capacity solutionproximal to the reference electrode.

Reference system for an ion selective sensor, for example anelectrochemical sensor, a glass electrode, a potentiometric sensorand/or the like. The reference system comprises a reference electrodecoupled with a redox active species, wherein the reference electrode isconfigured in use to contact a low buffer/low ionic strength analyte,such that the redox active species controls the pH of the environmentlocal to the surface of the electrode. In embodiments of the presentdisclosure, the voltammetric response of the reference electrode, whenin contact with a low buffer/low ionic strength analyte, to an appliedvoltammetric sweep includes a peak or a trough corresponding the pH ofthe local environment set by the redox active species. Since, the localenvironment pH set by the redox active species will be constant for theparticular redox species and because the voltammetric response, thepotential of the peak and/or trough and/or other feature in thevoltammetric response, will also be the same for the particular redoxspecies, the reference system will produce an absolute/constantreference measurement that can be used as a reference for any form ofion selective sensor.

In chemistry, pH is a numeric scale used to specify the acidity orbasicity (alkalinity) of an aqueous solution. It is approximately thenegative of the logarithm to base 10 of the molar concentration,measured in units of moles per liter, of hydrogen ions. More preciselyit is the negative of the logarithm to base 10 of the activity of thehydrogen ion. Solutions with a pH less than 7 are acidic and solutionswith a pH greater than 7 are basic. Pure water is neutral, being neitheran acid nor a base.

pH measurements are important in agronomy, medicine, biology, chemistry,agriculture, forestry, food science, environmental science,oceanography, civil engineering, chemical engineering, nutrition, watertreatment and water purification, as well as many other applications.

For nearly a century, pH has most commonly been measured using a glasselectrode. The glass electrode is a combination electrode that combinesboth a glass and a reference electrode into one body. The combinationelectrode consists of the following parts: a sensing part of theelectrode, a bulb made from a specific glass; an internal electrode,usually silver chloride electrode or calomel electrode; an internalsolution, usually a pH=7 buffered solution of 0.1 mol/L KCl or 1×10⁻⁷mol/L HCl; a reference electrode, usually the same type with a referenceinternal solution, usually 0.1 mol/L KCl; a junction with studiedsolution, usually made from ceramics or capillary with quartz fiber; andthe body of electrode, made from non-conductive glass or plastics.

Glass electrodes cannot be used in many industries because of theirfragility, requirement of calibration before use and need to be storedunder appropriate conditions. In addition, it is well-known in the artthat the reference electrode of the glass electrode, normally asilver/silver chloride system, suffers from drift, such that themeasurements from the glass electrode are inaccurate. This issue ofdrift in reference electrode systems, including the silver/silverchloride reference electrode, is well-known and there is an unmet needin the ion selective/electrochemical sensor industry for a referencesystem that does not drift.

A number of chemical analysis tools are known from chemical laboratorypractice. Such known analysis tools include for example the varioustypes of chromatography, electrochemical and spectral analysis.Particularly, the potentiometric method has been widely used for themeasurements of water composition both in the laboratory and in thefield of ground water quality control. U.S. Pat. No. 5,223,117 disclosesa two-terminal voltammetric microsensor having an internal referenceusing molecular self-assembling to form a system in which the referenceelectrode and the indicator electrode are both on the sensor electrode.The reference molecule is described as a redox system that ispH-insensitive (no proton transfer), while the indicator molecule is pHsensitive and is formed by a hydroquinone based redox system having apotential that shifts with the pH. Both, reference molecule andindicator molecule layers are prepared by self-assembly on gold (Au)microelectrodes. In the known micro-sensor, a pH reading is derived frompeak readings of the voltammograms.

Recently there has been significant work in the development of pHsensors for use in the water industry, where the concentration ofdissolved buffer and/or ionic salt is low. Interest in this area stemmedfrom the work by Compton et al., (see C. Batchelor-McAuley, B. R. Kozub,D. Menshkau, R. G. Compton, Voltammetric Responses of Surface-Bound andSolution-Phase Anthraquinone Moieties in the Presence of UnbufferedAqueous Media, J. Phys. Chem. C 115, pp. 714-718 (2011)), who showed theuse of classical quinone/hydroquinone voltammetry to monitor pH in thesesystems failed. Compton established that the proton coupledelectrochemical process perturbed the pH of the solution locally to theelectrode when little or no buffer and/or ionic salt was in the analytesolution. Compton's work suggested the unsuitability of redox activespecies to measure pH in low buffer analytes.

As well as measuring pH in source water, there is also a need to measurepH in seawater and saline solutions. One important reason for measuringpH in saltwater is to monitor the effects of carbon dioxide in theatmosphere on the pH of the Oceans. As part of its operationaldefinition of the pH scale, the IUPAC defines a series of buffersolutions across a range of pH values (often denoted with NBS or NISTdesignation). These solutions have a relatively low ionic strength(˜0.1) compared to that of seawater (˜0.7), and, as a consequence, arenot recommended for use in characterizing the pH of seawater, since theionic strength differences cause changes in electrode potential. Toresolve this problem, an alternative series of buffers based onartificial seawater have been developed. This new series resolves theproblem of ionic strength differences between samples and the buffers,and the new pH scale is referred to as the ‘total scale’, often denotedas pHT. The total scale was defined using a medium containing sulfateions. These ions experience protonation, H⁺+SO₄ ²⁻⇄HSO₄ ⁻, such that thetotal scale includes the effect of both protons (free hydrogen ions) andhydrogen sulfate ions: [H⁺]T=[H⁺]F+[HSO₄ ⁻].

Recently there has been significant work in the development of pHsensors for use in the water industry where the concentration ofdissolved buffer and/or ionic salt is low. For purposes ofclarification, the water industry concerns the monitoring, management,transport and/or of water, which in general does not contain largeamounts of ionic salts or dissolved buffer. Traditionallyelectrochemical pH sensors have focused on either glass electrode orISFET technologies, which ultimately suffer from the same inherentissue, drift of the reference systems and thereby requiring constantrecalibration. Although a solution to the issue on the ISFET has beenproposed which uses a second reference electrode species in a controlledenvironment to account for drift in the primary electrode, the ISFETelectrode still needs to be stored under wet conditions and recalibratedafter each series of measurement for validation purposes.

Over the past 15 years, work has been performed on developing a pHsensor utilizing traditional hydroquinone/quinone chemistry as the pHsensing element and where reference electrode drift is overcome in thesensor using a species that is insensitive to pH, similar to themicrosensor described above. In all of this work, the examples of thespecies that are insensitive to pH that have been described/used undergoonly an electron transfer process, which limit the molecules that can beutilized. Despite this limitation, such insensitive species providesignificant advantages over the traditional reference techniques, as thereference systems using the pH insensitive species can be stored dry anddo not require a stable external reference electrode; hence the pHsensor using such a reference system can provide for calibrationlessoperation. The challenge is to find a stable reference compound capableof surviving deployment over a long period of time and/or beinginsensitive to varying concentrations of species/contamination foundwithin the solution/analyte being tested. To this end a significantamount of focus has been on the use of ferrocene based species, whichspecies undergo a one electron oxidation to the resulting ferriceniumion. Judicious manipulation of the ferrocene structure enhancesstability, however polymeric based ferrocene has shown to suffer fromelectron mobility issues through the ferrocene chain. In addition,several ferrocene molecules have been shown to have redox potentialssensitive to the concentration and type of anion species in thesolution/analyte being tested and hence can only be used as a referencewhen the anion system does not change greatly. In addition, ferrocenereference systems need to be calibrated in the local environment priorto deployment.

A problem for pH sensors, ion selective sensors, glass electrode sensorsand electrochemical sensors, is the need for a stable referenceelectrode. In the glass electrode described herein, the referenceelectrode in KCl solution can drift during operation making the probeinaccurate. With electrochemical sensors using redox systems, referencessimilar to those used in the glass electrode will also drift. Moreover,use of redox sensitive species that provide an electron, such asferrocene, as a reference are problematic because the ferrocene isdifficult to stabilize on the electrode and the redox potential of thespecies may drift because of the instability. To-date, the referenceelectrode has been a major issue with ion selective sensors,electrochemical sensors, glass electrodes and/or the like.Electrochemists often suggest using the power supply as a reference toavoid the issues discussed herein, but even the power source cannotprovide a stable reference. In addition, the current reference systems,such as calomel, silver-silver chloride systems and/or the like may notbe usable for certain applications as they are bulky, disposed in achamber behind a frit or the like, and so may not be suitable forsensors for use in confined spaces/locations, such asmedical/pharmaceutical sensors, microfluidics and/or the like.

Further embodiments of the present disclosure provide a calibrationsystem for an electrochemical sensor. More particularly, but not by wayof limitation, in some embodiments an electrode is provided that isconfigured to contact a fluid and control the pH of the localenvironment of the fluid proximal to the electrode, such that a responseof the electrode to an applied electrical signal is indicative of the pHof the local environment. In some instances, the fluid contacted by theelectrode comprises a reference solution of a reference system of theelectrochemical sensor.

Electrochemical sensors are constantly being developed to measure anddetect chemicals. Electrochemical sensors essentially fall into threecategories: potentiometric sensors, voltammetric sensors andamperometric sensors.

Potentiometric sensors are one of the most common types ofelectrochemical sensor. Potentiometric sensing is the basis for glasselectrodes, which are used for measuring pH, sodium (Na+), potassium(K+), lithium (Li+) and the like, solid membrane electrodes based on thechemical process AgX for X−, liquid membrane electrodes, e.g. electrodescontaining a ligand for M+ complexation and used in calcium (Ca+),potassium (K+) sensors and the like, pH-meter-based gas detectors, e.g.carbon dioxide (CO2) sensors and the like, ammonia (NH3) sensors etc.,and some solid oxide sensors, e.g. zirconia-based oxygen (O2) sensorsand the like. Potentiometric sensors measure a potential differencebetween an electrode or environment that is sensitive to the desiredanalyte and an electrode or environment that is insensitive to theanalyte. In such sensors, an electrode or environment that is sensitiveto the analyte is generally referred to as the sensing electrode and theelectrode or environment that is insensitive to the analyte is generallyreferred to as the reference electrode.

Ion-sensitive field-effect transistors (ISFETs) are a new generation ofsolid-state potentiometric sensor. In an ISFET, the sensing electrode isreplaced with ion selective field effect transmitter, which measures avoltage between a source and a drain that is dependent on aconcentration of an analyte in a solution being measured. To processproperties of an analyte this source-drain voltage is measured againstan output from a reference electrode housed in a well-definedenvironment.

For both traditional potentiometric and ISFET sensors, significant workhas been performed to developing novel sensing electrode to measuredifferent analytes/ions and/or to improve the accuracy/sensitivity forsensing of an ion/analyte. This work has resulted in the development ofa range of commercial sensors that can achieve the desired selectivityand sensitivity to measure a range of analytes/ions.

For purposes of this disclosure, potentiometric sensors, ISFET sensorsand/or the like are referred to as ion selective electrodes (ISEs) sincethe sensing electrode in the sensors is tuned to detect/measure aselected ion.

Amperometric sensors measure ion concentration by monitoring the changein current as a function of the analyte. The sensing chemistry orsubstrate is typically tuned to be selective to the analyte. The sensingelectrode is held at a fixed potential with respect to a referenceelectrode and the analyte (or a product formed from the adductsinteraction with the sensor) is oxidized or reduced. This oxidization orreduction changes the current providing for detection of a concentrationof the analyte present.

Voltammetric sensors measure ion concentration by measuring a currentresponse as a function of swept potential. Voltammetric sensors are lesswell-known than, not as commonly used as potentiometric and amperometricbased sensors; however voltammetric sensors can provide greaterinformation than single point sensors. In a potentiometric sensor, apotential is swept with respect to a reference electrode andvoltammetric peaks arising from oxidation of an analyte (or a productformed from the adducts interaction with the sensor) can be determinedfrom the potential at which the peaks are observed and/or the measuredcurrent. The peak data from a voltammetric sensor may be used fordetecting/measuring multiple analytes using the same sensor interface,as in metal ion analysis.

Reference electrodes are generally used in all three types ofelectrochemical sensor to provide a stable, drift-free, accurate valueof potential as a reference voltage against which a variable output fromthe sensor, varying with analyte concentration, is measured. Inpotentiometric/galvanostatic sensors, such as ISEs and the like, apotential across/between the working and reference electrodes ismeasured and the current is held at known value. In voltammetricsensors, the potential is swept between the working and referenceelectrodes and the current output at the working electrode is measured.For amperometric sensors the measured current is determined at thesensing electrode and the potential at the working electrode is heldagainst a reference electrode that is at a stable, set potential.

The standard hydrogen electrode is the reference for standard electrodepotentials. The hydrogen electrode contains a platinized platinumelectrode immersed in a solution of 1 molar hydrochloric acid (HCl)under a 1 atmosphere (atm) pressure of hydrogen. The hydrogen electrodeis extremely accurate, however it cannot be used in operational sensors.Therefore, alternatives have been developed, which are typically basedon metals in intimate contact with a sparingly soluble salt of thecorresponding cation. The table below highlights some common referenceelectrode half-cell reactions.

Standard Electrode Electrochemical Reaction Potential/V AgBr + e⁻ = Ag +Br⁻ 0.071 AgCl + e⁻ = Ag + Cl⁻ 0.222 HgO + H₂O + 2e⁻ = Hg + 2OH⁻ 0.098Hg₂Cl₂ + 2e⁻ = 2Hg + 2Cl⁻ 0.268 Hg₂SO₄ + 2e⁻ = 2Hg + SO₄ ²⁻ 0.613

The common electrode comprises a silver/silver chloride (Ag/AgCl) orcalomel electrode, which is held within a defined environment behind aporous frit that allows for electrical conductivity to the solution thatthe sensor is measuring. In all cases the activity of all species(metal, sparingly soluble salt) except one (the ion in the solution)should be essentially unity. This is to ensure the reference electrodeis stable. However, the porous nature of the salt bridge, which is usedto ensure electroneutrality between the reference electrode chamber andthe analyte containing solution, means that the properties of thespecies in the reference chamber can change and the reference potential,as a result, drifts. The salt bridge is porous to provide for electricalconductivity between the two chambers. In practice, a too highlyresistive salt bridge should and a too porous salt bridge should beavoided. The latter would allow faster transport of ions and thus alterthe conditions of the reference electrode chamber rapidly.

A common system is that of a silver/silver chloride reference system.The Ag/AgCl electrode system is stored in a solution with knownproperties when not being used to prevent the electrode drying out. TheAg/AgCl sensor requires periodic recalibration due to drifts in thereference electrode potential, as described above. Obviously, as areference system for an electrochemical sensor, it is essential foraccurate measurement by the sensor that the reference system produce aconstant reference potential. However, in practice the referencepotential may change because the chemistry of the electrode/solution inwhich the electrode is immersed may breakdown/change and/or becausesince the frit is porous solutions/analytes can enter the referencesystem and perturb the reference potential. Furthermore, potentiometricsystems typically operate in a continuous single point measurement mode,which is a mode where a known current is held between the reference andsensing electrode (typically 0) and the potential difference isconstantly measured and used as an analytical signal. Any changes in thereference system will perturb this measurement mode and cause a drift inthe potential difference/reference potential measured and thus affectthe accuracy of the measurement.

In use, the reference potential of a conventional reference systemdrifts when deployed in a solution to be measured due to diffusion ofsolution through the porous frit into the reference system.

Methods to overcome reference electrode changes/drift have involved, forexample, deploying two measurement devices to make separatemeasurements. The first of these two measurement device methods uses afast-responding potentiometric system in which the reference electrodeis known to drift, and the second method uses an optical measurement tocalibrate changes in the reference electrode system. However, the firstmethod requires a system with a fast response time and the second methodrequires an additional colorimetric system, which adds expense to thesensor.

Testing has shown that a potentiometric system provides a varying outputwhen deployed against a colorimetric system for the measurement of pH inoceans. It has been shown that there is a difference of 0.1 pH unitsbetween measured (uncorrected potentiometric) and corrected (against thecolorimetric system) potentiometric system output, for a pre-calibratedsensor. This difference in value arises dues to the changes in thereference electrode system of the potentiometric sensor duringdeployment and shows how erroneous results would have been presentedshould the colorimetric sensor not been present to recalibrate thesensor in-situ. This method of recalibration although efficient isexpensive and would not be suitable for all electrochemical sensorsystems, where a suitable colorimetric measurement system may not beavailable.

Drift in the reference electrode potential during use of anelectrochemical sensor is almost impossible to monitor without one ofthe systems described above, and as a result, the drift in the referencepotential may be wrongly assigned to variations in the analyteconcentration causing a loss in sensor accuracy. In general, referenceelectrode drift may be addressed by periodic re-calibration of theelectrochemical sensor, which may be performed before every use of thesensor or periodically. However, for long duration/autonomous deploymentof an electrochemical sensor without user intervention/calibration, thedrift may increase with time making sensor measurements inaccurate andeven meaningless. Corrections for reference electrode drift may be madeafter use of an electrochemical sensor by recalibrating theelectrochemical sensor after use, determining changes in the calibrationfactors before and after of the electrochemical sensor and using thechanges in the calibration factors to extrapolate drift-corrected datafrom the data measured by the electrochemical sensor. In general, linearextrapolation is used to correct the measured data.

Clearly, such extrapolation reduces the accuracy of the electrochemicalsensor and for long-duration measurement the accuracy is severelycompromised. Additionally, since drift is unlikely to be a lineareffect, linear extrapolation can often produce poor drift correction.For regulatory purposes, it is often necessary to calibrate sensorsusing a standard extrapolation process to ensure for standardizedmeasurements. This can mean that frequent calibration is required and/orthat inaccuracy is built into the data. More importantly, thecalibration requirement for existing reference systems means that thesensors require constant calibration, which may be expensive and orrequire user intervention, meaning the sensor cannot be accurately usedin an online process and/or autonomously.

Several researchers have taken on the challenge of increasing thestability of the reference electrode and numerous methods have beenproposed to overcome the issue.

U.S. Pat. No. 5,419,826 describes an ion-selective reference probeadapted for use with potentiometric measurement systems. The referenceprobe is non-chloride based and employs a specially adapted electrolyte,which is reversible with regard to ionic activity.

United States Patent Publication Number 20030024812 discloses a solidstate electrochemical reference system, containing two or moreelectrodes, wherein the half-cell potential of at least one electrode isdetermined by the concentration of a specific ion anticipated to bepresent in all test solutions. The ion concentration measured in thecell by a first electroanalytical technique does not depend on a knownreference electrode potential, such that said electrode, its half-cellpotential being calculable from the measured ion concentration, canserve as a reference electrode in one or more subsequentelectroanalytical techniques that do depend on a known referenceelectrode potential, said subsequent technique or techniques beingcarried out in the same cell.

U.S. Pat. No. 6,398,931 details an improved combination ion-selectiveelectrode apparatus comprising an electrode body, a reference electrode,and an ion-sensing electrode. The reference electrode comprises anion-permeable junction and a removable membrane cap contains anion-selective membrane. The membrane cap can be removed from theion-selective electrode apparatus without endangering the integrity ofthe reference electrode and is distinct from the ion-permeable junction.

European Patent Number 2 932 249 describes a reference electrode for anelectrochemical sensor that comprises an inner reference element, whichinner reference element has been embedded into a solid electrochemicallyactive composite material.

U.S. Pat. No. 7,462,267 describes a reference electrode consisting of ametal in contact with an electrolyte containing an anion or cation whoseconcentration in part determines the redox potential of the electrode.This electrolyte contains a polyelectrolyte that partially andreversibly binds the chemical cation or anion thus lowering the freeconcentration of the cation or anion compared to the osmotic pressure ofthe same concentration of cation or anion if present as a simple salt.The polyelectrolyte can be anionic or cationic depending on thechemistry of the redox electrode and a thickener may also be added tothe electrolyte.

However, to-date, the proposed techniques for addressing drift of thereference system of electrochemical sensors are complex, require fragilecomponents, require careful handling/operation of the electrochemicalsensor, require regular maintenance, require periodic calibration and/orthe like, and even using the proposed techniques the detrimental effectof reference system drift may only be masked or may even be compounded.

SUMMARY

In embodiments of the present invention, a redox active species iscoupled with a reference electrode and the reference electrode isconfigured to contact a low buffer and/or ionic strength analyte, suchthat the redox active species sets the pH of the environment local tothe surface of the reference electrode surface. In some embodiments, thereference electrode may comprise a “naked” electrode that is configuredfor use in a sensor that is configured to be used to measure propertiesof and/or in a low buffer/low ionic strength analyte. For example, thesensor may be a sensor designed to measure pH of water or seawater or tomeasure pH or presence of a particular ion in a low buffer/low ionicstrength analyte, such as for example a saline solution in a medicalsystem or the like. In other embodiments, the reference electrode may bedisposed behind a frit or the like in a chamber containing a lowbuffer/low ionic strength liquid so that the sensor to which thereference system is attached may be used to measure properties ofbuffered and/or high ionic strength analytes or the like.

By way of example, anthraquinone, a common redox active species forelectrochemical sensors, will set a pH of approximately 10 for the localenvironment of an electrode when the electrode is disposed in a lowbuffer and/or low ionic strength analyte and a reducing potentialapplied to the electrode. In preferred embodiments of the presentdisclosure, the redox active species is selected to provide a localenvironment pH for the reference electrode that is not 7, so that theresponse of the redox active species can be better identified in thevoltammetric response of the reference system (low buffer/low ionicstrength analytes such as water, seawater, saline solutions and/or thelike generally have a pH of 7). In preferred embodiments of the presentdisclosure, the redox active species may be selected to set a pH localto the reference electrode that is a least one pH unit greater or lowerthan pH 7. In some embodiments, the redox active species may comprise anacidic or an alkaline chemistry, such as salicylic acid or the like, andas such may set the pH of the environment local to the referenceelectrode at a distance of greater than one pH unit from pH 7. In someembodiments, a redox active species may be used in conjunction with achemistry that sets the pH of the low ionic strength/low buffer analytecontacting the reference electrode. For example, a redox active speciesmay be disposed under a layer of an acidic or alkaline chemistry thatsets the pH of the environment local to the reference electrode in thelow buffer/low ionic strength analyte. In this way, an acidic oralkaline chemistry that sets the pH of the local environment of thereference electrode greater than one pH unit from 7 may be used with astable redox species that produces a strong voltammetric response.

Previously, in electrochemical sensing, as noted by Compton et al., thefact that a redox species controls the pH of the environment local to anelectrode was seen as detrimental, since the redox species in such asystem could not be used to measure the actual pH of the analyte.However, it was realized by the inventors of the present system that theeffect of the redox active species controlling the pH of the localenvironment could be used in a reference system, since the pH of thelocal environment of the electrode when contacted with a low bufferand/or low ionic strength analyte is an absolute determined by thechemistry of the redox active species and the response of the redoxactive species that pH is also an absolute again set by the chemistry ofthe redox active species, where the redox active species will produce avoltammetric response to a certain pH that is defined by the chemistryof the redox active species. Sensor configurations have always tried toprevent the reference electrode material controlling/changing the pH ofthe local environment as this was seen as detrimental to the sensingprocess. However, applicants have found that reference species that cancontrol the local environment, and in particular the pH of the referenceenvironment, can facilitate the use of pH active and redox activecompounds as reference species by using the correct functionalisationand having an understanding of the environment in which the sensor willbe immersed.

In some embodiments, the redox active species (sometimes referred toherein as the reference species for conciseness) is configured tocontrol the local environment under certain conditions, where theseconditions are provided by the analyte being sensed by the sensor. Forexample, acidic or alkaline reference species can be used that controlthe local environment of the reference electrode system when theelectrode is being used in water, seawater, saline solutions or thelike, where low ionic strength, low natural buffer potential or the likeof the analyte provides for the reference species controlling the localenvironment, where this will not occur with other analytes. Inwater/seawater/saline solutions or the like, because of the low ionicstrength and/or low natural buffer potential, the acidic/alkalineproperties of the reference chemistry can be used to set the local pHfor the reference electrode to greater than one pH unit from pH7. If,however, the ionic strength and/or natural buffer potential increases inthe analyte, water and/or seawater, the pH of the analyte, water and/orseawater will control the environment local to the reference electrode.In such aspects, because the pH properties of the reference species areknown, the reference can be used to determine ionic strength and/ornatural buffer of the analyte.

In other embodiments of the present disclosure, a calibration system isprovided that comprises a calibration electrode, which may comprise aworking electrode, that produces a voltammetric electrochemicalmeasurement that is used to verify/calibrate/adjust the referencepotential of a reference electrode of an electrochemical sensor.

In embodiments of the present disclosure, the voltammetricelectrochemical measurement is used to calibrate/adjust the referencepotential to remove/mitigate drift in the reference electrode. In thisdisclosure, the term “calibrate” is used to describe adjusting areference potential of a reference system to account for/remove driftand/or changes in properties of the reference system, such as thereference solution and/or the reference electrode.

The calibration electrode is configured in use to contact a solution andto set the pH in the solution local to the surface of the calibrationelectrode. In some embodiments, the calibration electrode can bedisposed in a low buffering capacity solution and/or a referencesolution of a reference electrode cell/chamber, as described herein. Thecalibration electrode comprises an electrode with an active surface thatis configured to control the pH of the low buffering capacity solutionand/or the reference solution proximal to the working electrode.

The active surface comprises redox functionalities that undergooxidation/reduction when an electrical signal is applied to thecalibration electrode. The redox functionalities produce an electricalresponse a redox potential, redox current to the electrical signal thatdepends upon the pH of the reference solution with which the activesurface is in contact. More particularly, the response of the redoxfunctionalities depends upon the pH of the local environment of thereference solution proximal to the redox functionalities. Merely by wayof example, for some embodiments of the present disclosure, redoxfunctionalities may comprise: quinone, derivatives of quinone,anthraquinone, derivatives of anthraquinone, salicylic acid, derivativesof salicylic acid, nitro groups (nitrobenzene and derivatives), aminogroups (amino benzene and derivatives), phenylenediamine based dyes(diphenyl-p-phenylenediamine and derivatives), phenpthiazine based dyes(methylene blue and derivatives), meldola blue, polyanilines,polypyroles, Tetracyanoquinodimethane. In some embodiments, Prussianblue, ferrocene, polyferrocenes, phthalocyanines and/or the like may beused.

In embodiments of the present disclosure, a voltammetric signal or anelectrode potential is applied to the calibration electrode. In suchembodiments, when the voltammetric signal is applied to the calibrationelectrode a voltammetric response is generated, where the voltammetricresponse is produced as result of the pH is the local environment, whichis set by the redox species/active surface of the calibration electrode,i.e., is a constant as the set pH produced by the redox species/activesurface does not vary. In some embodiments, the voltammetric responsemay be processed as a voltammogram comprising maxima and/or minimacorresponding to a peak oxidation current(s) and peak reductioncurrent(s), respectively. The peaks in the redox current occur atpotentials that depend upon the pH set by the active surface.

In embodiments of the present disclosure, the active surface, because ofits chemistry/properties, sets a particular pH proximal to thecalibration electrode. In some embodiments, the redox functionalities ofthe active surface may set the local pH. In some embodiments, the redoxfunctionalities in combination with other functionalities of the activesurface may set the local pH. In some embodiments, the otherfunctionalities may set the local pH.

As long as the chemistry/properties of the active surface does notchange, the pH of the local environment is constant. As such, a responseof the calibration electrode to an applied electrical signal, where theresponse is generated by the redox response of the redox functionalitiesto the applied signal in the presence of the set pH, is a constant. Forexample, where the applied signal comprises a voltammetric signal, avoltammogram of the response will include peak potentials correspondingto peak oxidation/reduction currents, with a constant peak potentialsince the set pH is constant.

In embodiments of the present disclosure, this constant responsegenerated by the calibration electrode to an applied voltammetric signalis used to calibrate a reference electrode of a potentiometric sensor.In some embodiments, a reference potential of the reference electrode ismeasured and compared to a peak potential from the calibrationelectrode, and the result of the comparison is used as a baselinemeasurement. This baseline measurement is used to calibrate thereference electrode when a comparison between the reference potentialand the peak potential differs from the baseline measurement, i.e., asthe reference electrode's reference voltage drifts.

In some embodiments of the present disclosure, potentials other than thepeak potentials are used for calibrating the reference electrode. Forexample, the voltammogram comprises turning points, locations ofgreatest change/slope, locations of least change slope, and/or the likethat are characteristic of the voltammogram resulting from the set pHand potentials of these locations may also be used or used in additionto the maximum potentials.

In some embodiments, the calibration electrode may be disposed insidethe reference cell/chamber of an electrochemical sensor. In suchembodiments, when an electrode potential is applied to the calibrationelectrode, the electrode potential will generate a redox current throughthe reference solution in the reference cell/chamber. In someembodiments, the electrode potential may be swept between thecalibration electrode and the reference electrode in the referencecell/chamber and current between the calibration electrode and acounter/or auxiliary electrode may be measured. In some embodiments, theelectrode potential may be swept between the calibration electrode andthe reference electrode in the reference cell/chamber, where when thecurrent is suitably low, the reference electrode can as the counterelectrode as well. In some instances, a potential of a peak value of theredox current is measured with respect to the reference electrode beingused in the potentiometric sensor.

The potential of the peak value depends upon the pH set by the activesurface and not on the bulk conditions of the reference solution.Through tracking variations in this measured potential with respect to apotential taken at the onset of deployment, variations in the referenceelectrode used for the potentiometric measurement will be recorded.Thus, any drifts or changes in reference electrode potential will beknown and can be used to recalibrate and modify the data provided fromthe potentiometric sensor in real-time.

In some embodiments, a processor may control applying electrical signalsto the calibration electrode and may use the calibration electroderesponse to calibrate the reference electrode. In some embodiments, theprocessor may apply electrical signals to the calibration electrode atthe same time or proximal to measurements made by the electrochemicalsensor to calibrate each sensor measurement. In some other embodiments,the processor may periodically apply electrical signals to thecalibration electrode to periodically calibrate the reference electrode.In such embodiments, calibration may defend on the period theelectrochemical sensor is to be used for, the number of measurements tobe made, the frequency of measurements and/or the like.

In some embodiments, the electrochemical sensor comprises a referencesystem comprising a reference electrode disposed in a solution in aliquid chamber, where the reference electrode is disposed behind aporous frit. The reference system may further comprise a bridging systemto maintain neutrality in the reference system. In some embodiments ofthe present disclosure, the calibration electrode is disposed in thesolution in the reference system. The solution comprises a referencesolution and the active surface of the calibration electrode sets the pHof the reference solution local to the calibration electrode. Byapplying a voltammetric signal to the calibration electrode avoltammogram is produced with features, such as peak potentials,determined by the local pH. In some embodiments of the presentdisclosure, a processor or the like compares the potentials of featuresin a voltammogram produced by the calibration electrode and corrects thereference potential of the reference system for any drift between thereference potential and the potential(s) from the voltammogram.

In some embodiments, other features of the voltammogram produced by thecalibration electrode may be used to determine the status/health of thereference electrode. For example, breakdown/changes in chemistry of thesolution in the reference system and or the reference electrode maycause changes in the voltammogram produced by the calibration electrode.These changes may include, creation of additional peaks in thevoltammogram, changes in overall shape of the voltammogram and/or thelike. Detection of these changes can be used to determine when thereference system is no longer functioning properly and can becommunicated to a user of the ion selective sensor or generate analarm/warning.

In some embodiments, calibration of the reference electrode potential isprovided by measuring a potential difference between the potential ofthe reference electrode and a potential of the calibration electrode,which potential is determined by applying a voltammetric sweep betweenthe calibration electrode and the reference electrode, potential ofcalibration electrode equals attributes of the redox wave which caninclude the peak maxima onset, half peak height of the voltammetric waveof the eredox active compound. The reference potentialism the potentialoff the silver\silver chloride couple min contact with the referencesolution. where any changes in the potential difference is used tocorrect/calibrate the output from the sensor. From the potentiometricsweep a characteristic of the sweep—which may be a peak in thevoltammetry, a change in direction of the sweep, a maximum rate ofchange on the sweep or other characteristic that can be ascertained bysignal processing—may be used to obtain a potential of the calibrationelectrode and this potential is used to calibrate the referenceelectrode. Since the voltammetric electrochemical measurement is not apotentiometric measurement, unlike the measurement from the referenceelectrode, the measurement provides a truly independent calibration.Moreover, the voltammetric electrochemical measurement may be madeperiodically reducing issues/maintenance requirements associated withcontinuous/high frequency measurements. Also, the calibration system maybe used with a robust reference system, such as an Ag/AgCl referencesystem and does not rely on potentiometrically measuring the presence ofan ion.

In embodiments of the present disclosure, the calibration electrodecomprises a redox species that controls the local environment proximalto the electrode. This control of the local environment may in someembodiments be provided by contacting the electrode with a lowbuffer/low ionic strength analyte, such as water, seawater or the like.In such, an environment, the calibration electrode, because of the lowbuffer/ionic strength of the analyte, ‘sees’ an environment controlledby the redox species itself. For example, a common redox species forelectrochemical sensors, anthraquinone will measure a pH of about 10 or11 when a voltammetric signal is applied because the anthraquinone willconsume protons during reduction triggering a change in the localenvironment that is measured by the sensor. This effect of the redoxspecies will occur for most redox species when the analyte contactedwith the redox species is a low buffer/low ionic strength analyte

In other embodiments, a redox species containing acid groups, suchsalicylic acid etc., or alkali groups, such as species containing aminegroups, acid groups will create an acidic or alkaline local environmentirrespective of the acidity/alkalinity of the fluid being sensed. Insuch embodiments, the local environment is controlled by theacidic/alkaline redox species even if the buffer/ionic strength of theanalyte contacting the redox species is not low. In some embodiments ofthe present disclosure, redox species with acid or alkali groups areused to move the pH of the local environment away from a neutralreading, a pH of 7, to provide a known reference potential output fromthe calibration electrode when a voltammetric signal is applied to thecalibration electrode.

Embodiments of the present disclosure may be used for allelectrochemical sensors, which require a stable, drift free referenceelectrode system. For example, potassium ion sensors utilize avalinomycin modified membrane to provide the ion-selective response, inconjunction with a standard Ag/AgCl electrode. The longevity of suchsystems is often compromised by instability in the reference electrode.In such systems, the addition of a calibration sweep system using anelectrode with a controlled environment would obviate the lifetimeissues associated with the drift in the reference electrode. Similarly,glass electrodes require constant calibration, in water managementoperations, this calibration is provided, in general, at least twice amonth. Some embodiments of the present disclosure can be configured toreplace this manual calibration.

Up until now, the effect of the redox species controlling the localenvironment has been identified as a weakness in electrochemical sensoroperation as it produces incorrect output from the sensor since thesensor measures properties of the local environment, which is controlledby the redox species, not the properties of the solution being tested.However as described herein, the effect provides an electrode that has aknown output, due to its control of the local environment, that may beused for calibration.

In sensors designed for use in low buffer/low ionic strength solutions,such as water/seawater or the like, the calibration electrode may becontacted directly with the low buffer/low ionic strength fluid as theredox species/active surface of the calibration electrode can controlthe pH of the local environment to produce a known/stable potentialoutput from the calibration electrode. In sensors that may be used withfluids with unknown properties and/or high ionic strength/bufferstrength, the calibration electrode may be contacted with a knownsolution, such as a reference solution kept behind a frit or the like,for example an aqueous solution with low ionic/buffer strength. In someother embodiments, the redox species/active surface of the calibrationelectrode may be tailored to control the local pH of the referencesolution. In such embodiments, the calibration electrode may becontacted with the same fluid environment as the reference electrode,i.e., a reference solution held in a reservoir behind a frit that allowsfor electrical/ion conductivity with the solution being tested/analyzed.

In some embodiments, the calibration system may comprise an additionalelectrochemical cell/chamber that is placed inside an existing referenceelectrode chamber. In such an arrangement, the reference electrode inthe existing reference electrode chamber may be used as a referenceelectrode for the calibration system.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, similar components and/or features may have the samereference label. Further, various components of the same type may bedistinguished by following the reference label by a dash and a secondlabel that distinguishes among the similar components. If only the firstreference label is used in the specification, the description isapplicable to any one of the similar components having the same firstreference label irrespective of the second reference label.

FIG. 1 shows, in general, a number of related sensor systems comprisinga sensor and reference electrode set-up. In detail the systems show in:FIG. 1A) The redox active species may comprise a regular redox activespecies such as anthraquinone or the like or may be a redox activespecies that includes directly attached moieties to control the localenvironment.

FIG. 1B) The reference electrode system comprises a dual layer in whichthe redox active species is separated/independent from a furtherchemical species that is used to control the local environment of thereference electrode. FIG. 1C) The redox active/pH active species usedwithin the reference electrode system by virtue of its oxidation orreduction properties can control the pH local to the surface of thereference electrode through the release/loss or gain of protons.

FIG. 2 : An electrochemical sensor comprising a reference electrodesystem in accordance with the present disclosure held with a potassiumchloride (KCl) solution and electrically connected to the analytesolution through a frit, wherein the reference electrode system is usedas an additional working electrode combined in a three electrode set-up.

FIG. 3 : An electrochemical sensor comprising a reference electrode inaccordance with the present disclosure held with a potassium chloride(KCl) solution where the reference electrode is placed behind a frit andused as a classical reference electrode in a three electrodeelectrochemical system.

FIG. 4 : A flow-type illustration of operation of a reference electrodesystem in accordance with the present disclosure.

FIG. 5 : Response of an electropolymerised salicylic acid electrode whenplaced in various pH adjusted sea water solutions.

FIG. 6 : A plot of peak potential with time, showing little variation inthe peak potential upon repetitive scanning.

FIG. 7 : A plot of peak potential as a function of scan number for aredox active process where the electrochemical process controls thelocal environment to achieve a reference potential.

FIG. 8 illustrates a glass electrode pH sensor with a referenceelectrode.

FIG. 9 illustrates an electrochemical sensor system with a calibrationsystem, in accordance with some embodiments of the present disclosure.

FIG. 10 depicts a schematic representation of a sensor output from anelectrochemical sensor comprising a calibration system, in accordancewith some embodiments of the present disclosure, using variousvoltammetric sweep profiles.

FIG. 11 illustrates an electrochemical sensor comprising a calibrationsystem, according to some embodiments of the present disclosure.

FIG. 12 depicts a schematic representation of a sensor output from anelectrochemical sensor comprising a calibration system, in accordancewith some embodiments of the present disclosure.

DESCRIPTION

The ensuing description provides some embodiment(s) of the invention,and is not intended to limit the scope, applicability or configurationof the invention or inventions. Various changes may be made in thefunction and arrangement of elements without departing from the scope ofthe invention as set forth herein. Some embodiments maybe practicedwithout all the specific details. For example, circuits may be shown inblock diagrams in order not to obscure the embodiments in unnecessarydetail. In other instances, well-known circuits, processes, algorithms,structures and techniques may be shown without unnecessary detail inorder to avoid obscuring the embodiments.

Some embodiments may be described as a process which is depicted as aflowchart, a flow diagram, a data flow diagram, a structure diagram, ora block diagram. Although a flowchart may describe the operations as asequential process, many of the operations can be performed in parallelor concurrently. In addition, the order of the operations may bere-arranged. A process is terminated when its operations are completed,but could have additional steps not included in the figure and may startor end at any step or block. A process may correspond to a method, afunction, a procedure, a subroutine, a subprogram, etc. When a processcorresponds to a function, its termination corresponds to a return ofthe function to the calling function or the main function.

Moreover, as disclosed herein, the term “storage medium” may representone or more devices for storing data, including read only memory (ROM),random access memory (RAM), magnetic RAM, core memory, magnetic diskstorage mediums, optical storage mediums, flash memory devices and/orother machine readable mediums for storing information. The term“computer-readable medium” includes, but is not limited to portable orfixed storage devices, optical storage devices, wireless channels andvarious other mediums capable of storing, containing or carryinginstruction(s) and/or data.

Furthermore, embodiments may be implemented by hardware, software,firmware, middleware, microcode, hardware description languages or anycombination thereof. When implemented in software, firmware, middlewareor microcode, the program code or code segments to perform the necessarytasks may be stored in a machine readable medium such as storage medium.A processor(s) may perform the necessary tasks. A code segment mayrepresent a procedure, a function, a subprogram, a program, a routine, asubroutine, a module, a software package, a class or any combination ofinstructions, data structures or program statements. A code segment maybe coupled to another code segment or a hardware circuit by passingand/or receiving information, data, arguments, parameters or memorycontents. Information, arguments, parameters, data, etc. may be passed,forwarded or transmitted via any suitable means including memorysharing, message passing, token passing, network transmission, etc.

Reference will now be made in detail to embodiments, examples of whichare illustrated in the accompanying drawings and figures. In thefollowing detailed description, numerous specific details are set forthin order to provide a thorough understanding of the subject matterherein. However, it will be apparent to one of ordinary skill in the artthat the subject matter may be practiced without these specific details.In other instances, well known methods, procedures, components, andsystems have not been described in detail so as not to unnecessarilyobscure features of the embodiments. In the following description, itshould be understood that features of one embodiment may be used incombination with features from another embodiment where the features ofthe different embodiment are not incompatible.

It will also be understood that, although the terms first, second, etc.may be used herein to describe various elements, these elements shouldnot be limited by these terms. These terms are only used to distinguishone element from another. For example, a first object or step could betermed a second object or step, and, similarly, a second object or stepcould be termed a first object or step. The first object or step, andthe second object or step, are both objects or steps, respectively, butthey are not to be considered the same object or step.

The terminology used in the description of the disclosure herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the subject matter. As used in thisdescription and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise. It will also be understood that theterm “and/or” as used herein refers to and encompasses any and allpossible combinations of one or more of the associated listed items. Itwill be further understood that the terms “includes,” “including,”“comprises,” and/or “comprising,” when used in this specification,specify the presence of stated features, integers, steps, operations,elements, and/or components, but do not preclude the presence oraddition of one or more other features, integers, steps, operations,elements, components, and/or groups thereof.

As used herein, the term “if” may be construed to mean “when” or “upon”or “in response to determining” or “in response to detecting”, dependingon the context. Similarly, the phrase “if it is determined” or “if [astated condition or event] is detected” may be construed to mean “upondetermining” or “in response to determining” or “upon detecting [thestated condition or event]” or “in response to detecting [the statedcondition or event],” depending on the context.

Embodiments of the present disclosure provide a reference electrodesystem comprising a redox active species that sets the pH of a lowbuffer/low ionic strength analyte local to the reference electrodesurface. A voltammetric response from the redox active species willinclude features/singularities corresponding to the set pH of the localenvironment of the reference electrode. These features/singularities inthe voltammetric response can be used as a reference for an ionselective/electrochemical sensor. For example, a redox active specieswill produce a peak redox current at a potential that corresponds to thepH of the local environment of the reference electrode, and thispotential can be used as a reference against which measurements from anion selective/electrochemical sensor can be made.

In embodiments of the present disclosure, the reference electrode iseither configured for use in a low buffer/low ionic strength analyte oris disposed behind a frit or the like in a solution of a low buffer/lowionic strength liquid. A voltammetric sweep is applied to the referenceelectrode and a voltammetric response of the reference electrode isprocessed. The voltammetric response will include features/singularitieswith properties that depend upon the pH of the local environment of thereference electrode, and because this pH is set by the redox species thevoltammetry response provides a reference from which measurements can bemade. In some embodiments, signal processing or the like may be used tomeasure the properties of the voltammetric response. Advantageously,because the location of the features/singularities can be predictedbased upon the pH set by the redox active species and the response ofthe redox active species to the set pH, weightings can be given toexpected locations of features/singularities and used in the signalprocessing. Additionally, in some embodiments, the voltammetric sweepmay be tailored to sweep potentials around expected locations offeatures/singularities in the voltammetric response.

In embodiments of the present disclosure, the reference system may beused in a sensor configured to measure properties of or detect ions inwater, seawater, saline solutions and/or the like having a lowbuffer/low ionic strength. In other sensors, the reference electrode maybe disposed in a reference solution of the low ionic strength/low buffersolution. Advantageously, the voltammetric response of the referenceelectrode may be used to identify issues with the reference system. Forexample, appearance of additional features in the sweep may identifycontamination/break down of the solution contacting the referenceelectrode. Large movement of features/singularities in the voltammetricresponse may also correspond to contamination of the solution contactingthe reference electrode or breakdown, loss of the redox active species.In reference systems where the reference electrode is directly incontact with the analyte being tested, changes in the location offeatures/singularities in the voltammetric response may be used todetermine a buffer level/ionic strength of the analyte.

Embodiments of the present disclosure may use the following redox activechemical structures (shown below) for the reference electrode, in whichthe redox active component molecule comprises carboxylic, sulfonicand/or amino moieties, in conditions in which the buffer concentrationwithin the analyte media does not overwhelm the concentration ofacid/base moieties of the redox active component molecule local to thesurface; such that the pH of the reference electrode surface is that ofthe pKa of the moiety.

In certain cases, such as that of salicylic acid (top left structureabove), a redox active-pH active polymeric layer can be formedcontaining carboxylic acid moieties. In certain cases where the bufferconcentration of the analyte being tested/sensed does not overwhelmthese moieties, the redox active components of the polymer only observesa pH local to the reference system consistent with that of the pKa ofthe molecule/moieties. Indeed, in certain embodiments of the presentdisclosure, as described below, a second reference system can be used toprovide a qualitative idea of the buffer concentration and a thirdelectrode can be used to determine the pH of the low salinity and/or lowbuffered media.

While the structures described above illustrate redox active specieswith “express” acidic or alkaline structures, redox active species suchas anthraquinone or derivatives thereof have been found to also set thepH of the local environment of a reference electrode when the electrodewas immersed in a low buffer/low ionic strength analyte. Thisillustrates the versatility of embodiments of the present disclosure,since anthraquinone and derivatives thereof are preferred redox activespecies because of their stability and consistent/regular and welldefined redox response.

FIGS. 1(A)-(C) illustrate an electrochemical pH sensor comprising areference system in accordance with embodiments of the presentdisclosure. The reference system is configured for use in a lowbuffer/low ionic strength analyte media.

In FIGS. 1(A)-(c), the system is run using a conventionalelectrochemical set-up with external reference and counter electrodesand incorporates the new reference electrode system and a pH sensingelectrode. Upon application of a potential ramp, the redox moiety of thereference system will be oxidized and reduced and show a potentialconsistent with the controlled local environment, whereas the pHsensitive electrode will provide a potential consistent with the pH ofthe analyte. The difference in the redox potential of the new referencesystem and the pH sensing electrode provides the pH of the solution,independent of any drift from the external electrochemical referenceelectrode.

In certain embodiments, the reference electrode system may comprise asingle layer in which a redox active species is configured to controlthe pH of the local environment of the reference electrode. The redoxactive species may comprise a regular redox active species such asanthraquinone or the like or may be a redox active species that includesdirectly attached moieties to control the local environment (FIG. 1(A)).In some embodiments, the reference electrode system may comprise a duallayer in which the redox active species is separated/independent from afurther chemical species that is used to control the local environmentof the reference electrode (FIG. 1(B)). In other embodiments, the redoxactive/pH active species used within the reference electrode system byvirtue of its oxidation or reduction properties can control the pH localto the surface of the reference electrode through the release/loss orgain of protons (FIG. 1(C)), i.e., the redox active species itself actsas the chemistry that sets the pH local environment of the referenceelectrode.

In FIGS. 1(A) through (C), the reference electrode is described as partof a pH sensor. This has been done merely by way of example as thereference system may be used as part of other types of ionselective/electrochemical sensors. For example, a sensor forelectrochemically measuring the presence of an ion in a saline solution,such as for example potassium, nitrogen, sodium and/or the like, may usethe reference output from the reference system of the present disclosureas the baseline against which its output is measured. Advantageously,because the reference system of the present disclosure, when used in asensor being used in a low buffer/low ionic strength analyte media cancomprise a naked electrode without a frit of the like, the referencesystem can be used in microfluidic systems, in-vitro or extra-vitrosensors and/or the like.

The electrode may comprise a conducting/conductive substrate, whichsubstrate may comprise: graphene, carbon nanotubes, carbon, glassycarbon, graphite, diamond, boron doped diamond or the like. Theconducting/conductive substrate may comprise a wire, such as a carbon orgraphene wire. In some embodiments, a paste/mixture of the redox activespecies may be formed and coupled with the electrode. For example apaste/mixture of the redox active species formed with carbon may bedisposed in a cavity in the electrode. The paste/mixture may include abinder/epoxy to hold the mixture together. In some embodiments theelectrode may be produced by printing conductive inks containing theredox active pH sensing material onto a substrate using screen printed,pad printed, flexiographic or rotar gravure printed technologies.Graphene and/or carbon nanotubes have been found as good material forthe electrode substrate as they are strong, provide good/regularconductivity, provide for uniform polymerization of the redox activespecies onto the substrate and can be used in micro-type electrodesystems.

Advantageously, the active redox active species may be selected to beinsoluble in aqueous media, and therefore may be solvent cast onto theelectrode surface. However, in some embodiments of the presentdisclosure, the active redox active species may be included in a carbonpaste or a carbon epoxy, immobilized within an electrode using a carbonpress, screen-printed onto an electrode and/or the like. In fact, manyof the active redox species described herein may form versatile redoxpolymers that can be effectively applied to the reference electrode.Merely by way of example, operation of a pH sensor is described for usein ocean and source water environment. Of course, sensors for otheranalytes in different environments can be used with the referenceelectrode described herein, but ocean and/or source water environment isone of the most challenging environments for a reference electrodebecause the water/seawater may contain low natural buffer capacityand/or ionic salt. Indeed, in certain aspects of this invention,described below, a second reference system can be used to provide aqualitative idea of the buffer concentration of the analyte and a thirdelectrode can be used to determine the pH of the low salinity and/orbuffered media.

For the environments which have a high buffering capacity and hencewould overwhelm the ability of the redox active species to control thepH of the local environment of the reference electrode, the newreference electrode system described herein may be enclosed in asolution having a low ionic strength/low buffer. In some embodiments,the redox active reference electrode may have electrical contact to theanalyte media through a porous frit, similar to first used inconventional reference electrode systems.

FIG. 2 details an embodiment of this architecture in which the newreference electrode system is held within a KCl solution or the like(other low buffer/low ionic strength analytes such as saline solution orthe like may be used, but KCL was selected for the example, because itlike AgCl is a well-established reference analyte). This design offers adouble protection with the local environment resisting perturbations inthe bulk changes in the KCL through ion transport across the frit (i.e.it would take dehydration disrupt effective operation of the referenceelectrode). The system as shown FIG. 2 would be used in the same way asthe sensor system described in FIG. 1 , in terms of reference systemoperation. Essentially, the chemistry of the redox active species of thereference electrode sets the pH of the environment in the KCl solutionlocal to the reference electrode and the redox active species recordsthis local pH as a reference which is communicated to the sensorprocessor

The sensor of FIG. 2 may comprise any kind of ion selective orelectrochemical sensor. The reference is superior to and meets thelong-felt need in the ion selective/electrochemical sensor art, becausethe redox active species when in contact with the low buffer/low ionicstrength analyte media set a pH of the local environment of theelectrode, the pH corresponding to the chemical properties of the redoxactive material, which may contain acidic or alkaline moieties or theoxidation or reduction properties of the redox species (the ability totake up or donate protons).

In certain cases, the external reference electrode can be replaced withthe new reference system, as illustrated in FIG. 3 , where the redoxactive functionalised electrode is placed behind a frit. In this casethe redox active species is first oxidized/reduced in a controlledmanner to set up a redox potential at the surface of the electrodeconsistent with the ratio of oxidized to reduced species on the surface.This illustrated system can then be used as a conventional threeelectrode reference electrode system.

FIG. 4 illustrates a flow-type schematic of use of a reference electrodesystem in accordance with embodiments of the present disclosure in anelectrochemical or ion selective sensor.

In some embodiments of the present invention, a processor 500 may beused to control the operation of a calibration electrode 530 tocalibrate a reference electrode 525 of an electrochemical sensor 520.

In a calibration process, the processor 500 may receive a signal fromthe reference electrode 525 and/or the electrochemical sensor 520containing a value of a reference potential of the reference electrode525. The processor 500 may control and/or may be a part of a signalgenerator/processor 510. Merely by way of example, the signalgenerator/processor 510 may comprise a potentiostat or the like.

As described herein, the calibration electrode 530 and the referenceelectrode 525 may be disposed with a reference chamber of theelectrochemical sensor 520 and may be in contact with a referencesolution therein. In other embodiments, the calibration electrode 530may be in a separate reference chamber, in a bulk solution being sensedby the electrochemical sensor 520 and/or the like.

As described herein, the calibration electrode 530 comprises a redoxactive species 533 that is sensitive to a pH of a solution contactingthe calibration electrode 530. In some embodiments, the redox activespecies 533 may comprise an active surface of the calibration electrode530 that has been modified to provide a redox active functionality.Merely by way of example, in some embodiments, the redox active speciesmay comprise anthraquinone, an anthraquinone derivative, salicylic acid,a derivative of salicylic acid, a combination of anthraquinone andsalicylic acid and/or the like.

As described herein, the redox active species 533 is configured to set apH of a local environment of a solution contacting the calibrationelectrode 530. This may be provided by contacting the redox activespecies 533 with a solution having a low buffering capacity, such thatit cannot buffer the effect of the redox active species 533 on the localenvironment. For example, the low buffering capacity solution maycomprise water, seawater, a sodium chloride solution, a potassiumchloride solution and/or the like. The redox active species 533 may haveacidic or basic properties, may comprise acid or base moieties and/orthe like.

In some embodiments, the processor 500 may control the calibrationelectrode 530 to generate a voltammetric signal and to apply this signalto the calibration electrode 530. A response of the calibrationelectrode 530 to the applied voltammetric signal is communicated to theprocessor 500 and/or the signal generator/processor 510. The response ofthe calibration electrode 530 is generated by reduction and/or oxidationof the redox active species 533 in the presence of the solutioncontacting the calibration electrode 530. Since the pH of the solutionis set by the properties of the redox active species 533 and/or anyother chemical species on the surface of the calibration electrode 530,the response is characteristic of the set pH of the local environment ofthe solution proximal to the calibration electrode 530.

In some embodiments of the present disclosure, the processor 500processes the response to identify features in the response. Forexample, in some embodiments, the processor 500 may identify a potentialof a singularity/peak in the response, where the singularity/peakcorresponds to a maximum of the reduction or oxidation currentassociated with the redox active species 533.

In some embodiments of the present disclosure, the processor 500 mayprocess a difference between the reference potential and the potentialassociated with the feature in the response and may use this differenceto calibrate the reference potential. For example, the referencepotential of the electrochemical sensor 520 may be calibrated with thecalibration electrode 530 when the electrochemical sensor 520 isset-up/deployed. When the electrochemical sensor 520 is set-up deployed,the reference electrode will, in general, be calibrated to a setreference value. In some embodiments of the present disclosure, when theelectrochemical sensor 520 is set-up/deployed, the reference potentialof the electrochemical sensor 520 may be communicated to the processor500 and compared with a peak potential in the response of thecalibration electrode 530 to an applied voltammetric signal. Thisdifference, a calibrated difference, may be recorded, entered in adatabase and the processor 500 may periodically apply furthervoltammetric signals to the calibration electrode 530 to generatefurther peak potentials and may compare these peak potentials withreference potentials obtained from the reference electrode 525 and usethese comparisons and the recorded/stored calibration difference may beused to calibrate the reference electrode 525.

In the present disclosure control over the local environment can beachieved in two ways: passive control, where the chemical/immobilizedlayer sets the local environment, or electrochemical control, where thenature of the electrochemical reaction sets the environment local to thesurface. In the former case, stability of the local environment isachieved without the need for controlling the electrochemicalparameters. However, in certain circumstances the electrochemicalparameters are tuned to induce electropolymerisation of the surfaceactive molecule to enhance the chemical stability of the electrode. FIG.5 details the response of a salicylic modified electrode in the presenceof sea water and in sea water where the pH has been altered by theaddition of acid or base. In this system the salicylic acid iselectropolymerised prior to the scans by cycling the potential topotentials where the phenol monomer is oxidized. The redox wave of theresulting polymeric species is found to be stable, consistent with thesalicylic acid electrode imparting control over the local environment.The peak potential is set by the pKa of the acid moieties of themolecule. As can further be seen in FIG. 6 , which details a plot ofpeak potential with time, this system shows little variation in the peakpotential upon repetitive scanning. The observation of this stablesignal shows that the electrochemical parameters do not influence thelocal environment.

In the second case, where the local environment is controlled by theelectrochemical process, control is provided by the electrochemicalparameters. In certain embodiments, an electrode is modified with aquinone moiety, which can be electrochemically reduced and subsequentlyoxidized. During the electrochemical process, protons are consumed localto the environment and as such an alkaline pH is achieved within thediffusion layer of the electrode. Maintaining and/or achieving thisalkaline environment is attained by applying a reductive potential tothe electrode surface (with respect to the redox potential of thespecies) or repetitive scanning/cycling of the potential.

FIG. 7 details the plot of peak potential against time showing the peakpotential measured for 20 consecutive scans, when the electrode wasplaced in a pH 10 environment. With each cycle the peak potential islowered, until it stabilizes between scans 17-20. The data can beexplained as follows; the potential is lowered due to theelectrochemical process consuming protons at the electrode surface,after each scan the local pH becomes more alkaline as further protonsare consumed. The more alkaline the pH, the lower the redox potential ofthe redox active species for a pH active molecule. Between scans 17 and20 the local environment is in equilibrium with the electrochemicalprocess and a stable signal is observed. In a real device, control ofthe system can be achieved through monitoring the peak potential aftereach scan; as the peak potential converges with repetitive scanning thesystem will monitor the peak potential until a stable signal is observedacross a defined number of scans.

In the case outlined, repetitive scanning was achieved using square wavevoltammetry, however other forms of voltammetry: cyclic, linear sweep,normal pulse, differential pulse and elliptical can also be used tosweep the potential. In some systems fixed potential amperometry orfixed current potentiometry may be used to set the local environmentprior to the electrochemical sweeps.

The following description provides reference to use of a calibrationelectrode in accordance with the present disclosure with a glasselectrode. This combination is made merely as an example of theoperation of the calibration electrode, since glass electrodes areprobably the most ubiquitous of electrochemical sensors, and as personsof skill in the art will understand, the calibration electrode may beused in the same manner with any other electrochemical sensor using areference electrode/system.

Redox active functionalities are functionalities that may be oxidizedand reduced, and redox activity may refer to either or both of thoseprocesses.

Redox functionalities and/or redox-active functionalities comprisefunctionalities that are tailored to be sensitive to the presence orconcentration of an analyte in a solution. For example, when avoltammetric signal is applied to the tailored redox activefunctionality, a redox current/potential generated by the redox-activefunctionalities will depend upon presence or concentration of theanalyte in the solution. The solution may comprise of hydrogen ions andin such cases the redox active functionalities are sensitive to the pHof the solution. For most eletrochemical sensors, the redox activefunctionalities can undergo a reversible electrochemical redox reactiondependent upon the concentration of analyte (hydrogen ions for a pHmeter; other analytes for other analyte sensing devices). For aconventional pH sensor, such as the glass electrode, the hydrogen ionsassociate with the glass membrane causing a change in the measuredpotential at the electrode housed behind the glass membrane with respectto the reference. In the conventional glass pH electrode, in essence,two Ag/AgCl electrodes are used; one behind the pH sensitive glass andone behind a conventional frit. As such, in some embodiments, thereforethe calibration electrode described herein may be used to determineconditions in the chamber behind the glass membrane and/or to calibratethe Ag/AgCl electrode behind the glass membrane.

In a sample solution with an applied electrical potential, for example,where there is a high concentration of hydrogen ions present in a samplesolution, the redox reaction occurs at a lower potential. Conversely,where there is a low concentration of hydrogen ions present in a samplesolution, the redox reaction occurs at a higher potential. Therelationship between these characteristic potentials and the samplesolution pH is a function of the chemical identity of the redox activefunctionality. An algorithm converts electrical potential to pH value toprovide a means of determining the pH of an unknown sample.

For some embodiments, redox active functionalities may comprise pHsensitive redox active functionalites such as for example: anthraquinone(AQ), phenanthrenequinone (PAQ), N,N′-diphenyl-p-phenylenediamine(DPPD), anthracene, naphthaquinone, para-benzoquinone, diazo-containingcompounds, porphyrins, nicotinamides, including NADH, NAD andN-methylnicotinamide, quinone thiol, monoquaternizedN-alkyl-4,4′-bipyridinium, RuO, and Ni(OH).sub.2, and derivatives ofthose compounds; CO-sensitive ASMs: ferrocenyl ferraazetine disulfide;alkaline metal cation sensitive ASMs:1,1′-(1,4,10,13-tetraoxa-7,1-diazacyclooctadecane-7,16-diyl dimethyl),ferrocenyl thiol, other ferrocene derivatives containing covalentlyattached cryptands, and certain metal complexes withFe.sup.2+/Fe3.sup.+, Co2.sup.+/Co3.sup.+, Cu.sup.+/Cu2.sup.+, ferrocenylferraazetine and ferrocenyl cryptands,1-hydro-1′-(6(pyrrol-1-yl)hexyl-4,4′-bipyridiniumbis(hexafluoro-phosphate) and or the like. Further, redox activefunctionalities that may be activated on a surface of the calibrationelectrode such as C—O functionalities are described herein.

A reference electrode is an electrode that may be used to establish thepotential difference applied to a working electrode of anelectrochemical sensor. Generally, reference electrodes comprise a fixedchemical composition and, therefore generate a fixed electrochemicalpotential. This fixed electrochemical potential provides for measurementof the potential difference applied to the working electrode of theelectrochemical sensor. It is imperative that the composition of thereference electrode remains constant, and hence almost no current shouldbe passed through the reference electrode to prevent electrolysis. Toprevent passing current through the reference electrode, electrochemicalsensors generally comprise a counter electrode to complete the circuit.However, two-electrode electrochemical sensors may be used where theworking electrode is a microelectrode. This is possible because thecurrents passed at the electrode are small.

A working electrode of the electrochemical sensor is the electrode atwhich the electrochemical process for detecting an analyte of interestoccurs. In a sensor, the working electrode may be sensitive to one ormore analyte(s) in a test solution, or it may be chemically modifiedwith analyte sensitive species/materials or surface modified so that asurface of the electrode comprises redox active functionalities. Theelectrochemical response of the working electrode is measured after aperturbation is applied. The perturbation may comprise an electricsignal, voltammetric signal and/or the like, for example, application ofa potential difference to the working electrode induces electrontransfer to occur, and the resulting current at the working electrodecan be recorded as a function of the applied potential.

FIG. 8 illustrates a glass electrode pH sensor with a referenceelectrode. The glass electrode is essentially a combination electrode,which combines both a glass electrode and a reference electrodes. Theglass electrode consists of a sensing part of the glass electrode, oftenreferred to as the sensing electrode which comprises a bulb 1 made froma specific glass, an internal electrode 2 and an internal solution 3.Merely by way of example, the internal electrode may comprise a silverchloride electrode, a calomel electrode or the like. The internalsolution 3 may comprise a pH=7 buffered solution of 0.1 mol/L KCl for pHelectrodes, 0.1 mol/L XCl for pX electrodes or the like, where X is theion under determination. In a glass electrode using a silver chlorideelectrode, a small amount of AgCl precipitate 4 may form inside theglass electrode.

The reference system 5, often simply referred to as the referenceelectrode, comprises a reference electrode 9 immersed in a referenceinternal solution 6. The reference electrode may comprise a silverchloride electrode, a calomel electrode or the like. The referenceinternal solution 6 may comprise a salt such as a silver chloride,potassium chloride or the like. For example, the reference internalsolution may comprise 0.1 mol/L KCl or the like.

The glass electrode further comprises a junction 7 that provides forcommunication between the internal parts of the glass electrode and thesolution being analyzed by the glass electrode. The junction 7 maycomprise a ceramic, a capillary containing asbestos or quartz fiberand/or the like. The glass electrode comprises a housing 8 housing thecomponents of the glass electrode. The housing 8 may be made fromnon-conductive glass, plastic and/or the like.

The bottom of the glass electrode balloons out into a round thin glassbulb. The pH electrode is best thought of as a tube within a tube. Theinner tube contains an unchanging solution. Also inside the inner tubeis the cathode terminus of the reference probe. The anodic terminuswraps itself around the outside of the inner tube and ends with the samesort of reference probe as was on the inside of the inner tube. It isfilled with a reference solution and has contact with the solution onthe outside of the pH probe by way of a porous plug that serves as asalt bridge.

FIG. 9 illustrates a calibration electrode in accordance with someembodiments of the present disclosure. The calibration electrodecomprises an electrode body 20. The electrode body may comprise carbon,a metal, a metal ion and/or the like. Carbon based electrodes andmetallic compounds are commonly used in electrochemistry because oftheir good electrical conductivity. Carbon materials also have lowdensity, low thermal expansion, and low elasticity and are costeffective, readily available, and suitable for modification. Theelectrode body in some embodiments may comprise carbon black, graphite,glassy carbon, carbon fibers, carbon nanotubes, multi-walled carbonnanotubes, edge plane carbon, graphene and or the like. Grapheneprovides a strong electrode with good conductivity and advantageouschemical properties.

The electrode body may comprise a substrate or the like that isconnected to an electrical connection 27. The electrical connection 27may comprise a wire or the like that is configured to communicateelectrical signals to and from the calibration electrode.

The calibration electrode comprises an active surface 23. The activesurface 23 comprises a surface that is configured to both set the pH ofa low capacity buffer solution proximal to the active surface andrespond to the set pH proximal to the active surface.

It is known that carbon materials respond to pH without activation. Thisresponse is due to the presence of chemisorbed oxygen on the graphitesurface that cause the formation of C═O groups. Methods have beendeveloped to modify the carbon materials to improve or tune theirelectrocatalytic, redox, or ion-sensing properties. These methods ofactivating the carbon material to provide for the immobilization ofpH-sensing substances onto the active surface can be divided into fivemain groups: (1) chemical modification of the carbon surface with oxygenand/or nitrogen containing functional groups; (2) covalent bonding ofpH-sensing compounds onto the active surface initiated either bychemical or electrochemical activation; (3) physical adsorption of pHsensing compounds onto the active surface; (4) fabrication of filmelectrodes on the active surface; and (5) fabrication of carboncomposite electrodes where the surface of the carbon composite comprisespH sensing substances.

FIG. 10 illustrates the active surface 27 formed on the electrode body23, where the electrode body 23 comprises a carbon material. In someembodiments, the electrode body 23 and the active surface 27 comprise asingle substrate and the active surface 23 simply comprises a surface ofthe electrode body 27 that is configured in use to be in contact with alow buffering capacity solution and/or a reference solution in thereference chamber of the electrochemical sensor. Merely by way ofexample, the electrode body 27 may comprise a composite electrode andthe active surface 23 is simply a surface of the electrode body 27 thatis configured to be contacted with the low buffering capacity/referencesolution.

In some embodiments, the active surface 23 is configured to be contactedwith a low buffering capacity solution. Such solutions, because of lowproton transfer rates resulting from their low buffer capacity proximalto the active surface 23, comprise a pH value proximal to the activesurface that is set by the properties of the active surface. In someembodiments, the properties of the active surface 23 are configured sothat the active surface 23 sets a pH value of a reference solutionproximal to the active surface 23. For example, the reference solutionmay comprise KCl, HCl and/or another salt configured to work with thereference electrode material. In some embodiments, the active surface 23may have acidic or basic properties so as to set a local pH in thereference solution to a pH above or below a pH of seven (7). In someembodiments, redox active functionalities of the active surface 23 mayset the local pH of the low buffering capacity/reference solution.

In embodiments of the present disclosure, the active surface 27 may bemetallic Like carbon, a metal may be treated so that substances areformed on a surface of the metal and these substances may comprisesubstances that will undergo oxidation/reduction in the presence ofprotons/hydrogen ions such that their electrical response in theirpresence depends upon a concentration of hydrogen ions, and is thereforeindicative of pH. As with the case of a carbon electrode, the electrodebody 23 may comprise the metal and the active surface 27 may simplycomprise a surface of the electrode body 23 that is configured to becontacted with a low buffering capacity/reference solution.

In some embodiments, the active surface 27 may be formed by a metallicion disposed in the low buffering capacity solution, where the lowbuffering capacity solution is a saline solution and then metal ion mayproduce a redox couple in the saline solution which provides for redoxactivity at a surface of the electrode body 23.

Boron doped diamond has also been found to be capable of being used tomeasure pH of a solution. In some embodiments of the present disclosurethe active surface 27 may comprise boron doped diamond. When a currentis passed through the active surface 27 in the presence of the lowbuffering capacity/reference solution, a response of the active surface27 is determined by a hydrogen evolution reaction on the active surface27, where the reaction is provided by:2H⁺+2e→H₂and, as such is indicative of pH.

The active surface technologies described have been developed to providean active surface that can be used to measure pH without the need todeposit reagents onto the sensing electrode, i.e., the sensing electrodeitself comprises the redox active functionalities. Such technology hasbeen pursued as it removes the issue of coupling reagents with theelectrode and to increase the robustness of the sensing electrode. Assuch, a main drive of the technology has been to increase the redoxresponse of the active surface to increase sensitivity.

As with electrochemical pH sensors using reagents coupled with thesensing electrode, the active surface, like the reagents, will set thepH of a low buffering capacity solution in a region of the low bufferingcapacity solution proximal to the sensing electrode. Moreover,Applicants have found that the active surface, like the reagents coupledwith the sensing electrode, can be configured to set the pH local to theactive surface when the active surface is contacted with a referencesolution of a reference electrode system. As with electrochemical pHsensors using reagents, this effect has been seen as detrimental tousing active surface type electrodes to sense pH of low bufferingcapacity solutions, such as water, seawater, saline solutions and thelike. To avoid this issue, active surfaces have been developed with lowconcentrations of redox active functionalities so that the pH settingeffect of the functionalities is swamped by the pH of the low bufferingcapacity solution.

In embodiments of the present disclosure, the active surface 27 maycomprise a carbon, carbon derivative, boron doped diamond or the likecomprising redox active functionalities that are configured to set thepH proximal to the active surface 27 such that when an electronic signalis applied to the active surface 27 a redox response of the activesurface 27 is determined by/identifies the pH set by the redox activefunctionalities.

In embodiments of the present disclosure, the calibration electrode ischemically or physically activated to generate redox activefunctionalities at the active surface 27. These redox activefunctionalities are configured to control the local environment of theelectrode surface either directly through the introduction of acidic orbasic moieties or indirectly through the introduction of redox activemoieties, which when oxidized or reduced, take-up or release protons,thus locally changing the pH at the electrode surface.

It has recently shown that mechanical, laser, heat and chemicalactivation of carbon surfaces (BDD, graphite, carbon nanotube, graphene,glassy carbon, basal plane carbon and edge plane carbon in solid orprinted formats) produce electrochemical active sites on the surface ofthe carbon.

The carbon chemistry depicted above illustrates the types of functionalgroups that can be produced on a carbon surface. Research has shown thatcarbon surface functionalities can provide good pH responses across theentire pH range. In the presence of low buffering capacity media, suchas those found within an ion selective electrode reference chamber,which typically contain a known concentration of inert electrolyte (KCl,NaCl, NaClO₃ ⁻, Na₂SO₄ etc.), the pH sensing capabilities of thesesystems fail and rather than measuring the pH of the low bufferingcapacity media, a sensor comprising a sensing electrode including carbonsurface functionalities will measure the pH set by the carbon surfacefunctionalities. This is because the carbon surface functionalitiescontrol the pH local to the electrode surface, due to the intake (orrelease) of protons by the carbon surface functionalities, making thelocal environment acidic or alkali.

FIG. 11 illustrates an electrochemical sensor comprising a calibrationsystem, according to some embodiments of the present disclosure. In FIG.11 , a novel means of calibrating the electrochemical potential of thereference electrode is provided by the introduction of an additionalvoltammetric electrochemical measurement.

In FIG. 11 , a calibration electrode 43 is placed inside a referenceelectrode chamber of an electrochemical sensor. A reference electrodefor the electrochemical sensor, which is being calibrated, may also beused as a reference electrode for the calibration system, i.e., in thecalibration system a voltammetric response of the calibration electrodemay be processed against a sweep applied between the calibrationelectrode, the reference electrode and/or a counter electrode. Forexample, in some embodiments, the calibration system of the presentdisclosure may comprise a two or three electrode system. In someembodiments, the reference electrode of the calibration system maycomprise the reference electrode of the electrochemical sensor. The twoor three electrode system may be used in a voltammetric system toproduce a peak potential corresponding to a pH of a solution in contactwith the electrodes. Which pH is set by the properties of thecalibration electrode.

In some embodiment, prior to deployment of the electrochemical sensor, avoltammetric scan can be taken between the reference electrode and thecalibration electrode and a potentiometric measurement recoded by thesensor in a known solution. This initializing scan sets the parametersof the electrochemical sensor taking into account how the particularelectrochemical sensor is behaving. In essence, this is a one-timeinitial calibration for the particular electrochemical sensor. Forexample, in an ISE, the calibration takes into account how theion-selective layer responds with respect to the reference electrode,and the voltammetric sweep measures the potential of the referenceelectrode with respect to the calibration electrode.

In embodiments of the present disclosure, the calibration electrode ischemically or physically activated to generate redox activefunctionalities on the surface of the electrode. These redox activefunctionalities are configured to control the pH of the localenvironment of the electrode surface, either: (a) directly through theintroduction of acidic or basic moieties; or (b) indirectly through theintroduction of redox active moieties, which when oxidized or reduced,uptake or release proton thus locally changing the pH at the electrodesurface. In some embodiments, the redox active functionalities maycomprise a combination of those in group (a) and those in group (b).

In some embodiments, the calibration system comprises a counterelectrode. However, in some embodiments, when the calibration electrodepasses sufficiently low current, the counter electrode may not berequired.

In some embodiments of the present disclosure, the calibration electrodemay comprise an active surface produced by mechanical, laser, heatand/or chemical activation of a carbon surface, such as boron dopeddiamond, graphite, carbon nanotube, graphene, glassy carbon, basal planecarbon and edge plane carbon in solid or printed formats.

In embodiments of the present disclosure, the active surface sets thelocal pH of a low buffering capacity solution and measures this pH togenerate a calibration signal that is used to correct drift in thereference potential, this may be or is by means of measuring a voltageand then determining the pH by means of a calibration plot. Inembodiments of the present disclosure, the redox active functionalgroups present on the active surface produce pH local to the activesurface that is independent of the ion concentration within thereference electrode chamber.

In embodiments of the present disclosure, a potentiometer 46 or the likemay be used to apply a voltammetric signal to the electrochemical sensorand to the calibration system. The electrochemical sensor's response tothe voltammetric signal may be used to determine properties, such asconcentration, of the selected ion. The calibration electrodes responseto the voltammetric signal may be used as a calibration signal tocorrect for any drift in the reference potential of the electrochemicalsensor.

In embodiments of the present disclosure, the calibration system may beused periodically by running a voltammetric sweep against thecalibration electrode, which is housed in the reference system behind aporous frit. The response of the calibration electrode to an appliedvoltammetric signal may take the form of a voltammogram. In someembodiments, a potential of the redox active species, such as a peakpotential comprising of a peak in the reduction/oxidation current of theredox active species, is used to correct any drift occurring in thereference electrode. A frequency of voltammetric sweep may be selectedin accordance with the application for which the electrochemical sensoris being used. In some embodiments, the electrochemical sensor may berun using a modified bi-potentiostat system.

FIG. 12 depicts a schematic representation of a sensor output from anelectrochemical sensor comprising a calibration system, in accordancewith some embodiments of the present disclosure. In FIG. 12 , theelectrochemical sensor generates output A at times t₁ through t₁. Theoutputs correspond to a selected ion being measured and are determinedfrom a response of the sensing electrode determined using a referencepotential of the reference electrode.

Output B illustrates a comparison of the reference potential to apotential obtained from the calibration electrode in response to avoltammetric signal, such as a voltammetric sweep. The potentialobtained from the calibration electrode may comprise a potential of apeak oxidation/reduction current produced by the calibration electrode,the redox functionalities on the active surface of the calibrationelectrode. Since the calibration electrode is maintained in contact witha low buffering capacity solution in the reference system, thecalibration electrode sets the pH of the low buffering capacity solutionlocal to the calibration electrode and the potential of the peakreduction/oxidation currents will be constant despite any changes to thelow buffering capacity solution.

In general, electrochemical sensor's use reference systems that compriselow buffering capacity solutions such as KCl solution and/or the like sothe standard reference solution does not need to be changed for use withthe calibration system. While the potential of the oxidation/reductioncurrents is described for calibration, any reference point in thevoltammetric response of the calibration electrode may be used, such asturning points, point of maximum or minimum change in slope of thevoltammogram or the like. Further, signal processing methods suchwavelet interpretation and or the like may be used to identify referencepoints in the voltammogram and their corresponding potential for use forcalibration of the reference potential. Advantageously, the peakredox/reduction potential of the calibration electrode response may bedetermined by testing of the electrochemical sensor when built, whichmay be used for batches of the electrochemical sensor's, or calculatedempirically, and this data may be used in the voltammogram analysis. Insome embodiments, a potential for either the peak reduction or oxidationcurrent is used to correct the reference potential for drift.

In FIG. 12 , a position of the reference potential relative to a peakpotential in the voltammetric response of the calibration electrode attime t₄ is shown to have shifted with respect to the relative potentialsat t₁, t₂ and t₃. Without the calibration system of the presentdisclosure, this change in the reference potential would have producedan inaccurate measurement at t₄. With the calibration system of thepresent disclosure, however, the drift of the reference electrode can becorrected and the correct measurement of the selected ion processed.

In embodiments of the present disclosure, the calibration system can beused to provide QA/QC of the electrochemical sensor's reference system.As provided in FIG. 11 , the electrochemical sensor includes anintegrated electrochemical cell/chamber and a potentiostat, and thesystem uses a voltammetric signal from the potentiostat to produce asweep between the reference electrode and the calibration electrode inthe integrated electrochemical cell/chamber to generate a potential thatmay be used for QA/QC of the reference electrode during operation of thesensor. The sensor may in some embodiments run using a modifiedbi-potentiostat system. Such a set-up obviates the need for referencesensor calibration prior to deployment as is the case with currentcommercial electrochemical sensor's. In embodiments of the presentdisclosure, prior to deployment, the internal circuit can be measuredand this may be used to set the parameters for the reference system.

While the principles of the disclosure have been described above inconnection with specific apparatuses and methods, it is to be clearlyunderstood that this description is made only by way of example and notas limitation on the scope of the invention.

The invention claimed is:
 1. A method of operating a reference electrodecomprising a redox species sensitive to proton concentration, the methodcomprising: contacting a solution with the reference electrode in orderto generate a reference potential for an electrochemical sensor; using apotentiostat to apply a voltammetric signal to the reference electrode,wherein the reference electrode is one electrode in a three-electrodesystem in the electrochemical sensor; and using a processor to process avoltammogram from a voltammetric response of the three-electrode systemto the voltammetric signal and to process the reference potential fromthe voltammogram, wherein: the reference potential corresponds to amaxima or a minima in the voltammogram; and a local pH generated in thesolution by the redox species is used for at least one of: adjustingparameters of the voltammetric signal applied to the reference electrodeor processing the reference potential from the voltammogram.
 2. Themethod according to claim 1, further comprising: using the referencepotential to process a measurement from the electrochemical sensor. 3.The method according to claim 1, further comprising: using thevoltammetric response to determine when the reference electrode iseither malfunctioning or operating below a threshold performance level.4. The method according to claim 3, further comprising: selecting adifferent reference electrode and applying the voltammetric signal tothe different reference electrode when it is determined that thereference electrode of the three-electrode system is eithermalfunctioning or operating below the threshold performance level. 5.The method of claim 1, wherein the electrochemical sensor comprises atleast one of: a voltammetric pH sensor, an ion selective electrode, anoxidation-reduction potential sensor, and/or a glucose sensor.
 6. Themethod of claim 1, wherein the solution is a low buffering capacitysolution.
 7. The method of claim 1, wherein the redox species comprisesone of salicylic acid, anthraquinone, or an anthraquinone derivate. 8.The method of claim 1, wherein the voltammetric signal comprises acyclic voltammetric sweep.
 9. The method of claim 1, further comprisingusing the reference potential to calibrate a Ag/AgCl reference electrodeof the electrochemical sensor which does not form part of thethree-electrode system.